Pharmacopoeia Monograph Methods

PharmacopoeiaMonograph MethodsHPLC and UHPLC methods for Regulated Drug Analysis

Merck Millipore is a division of

ContentMolecular Structures 3

Applications Index 4

Introduction 5

Pharmacopoeial Requirements 6-14- Important Changes in USP 37 9-10- Case study – method transfer from particulate to monolithic column 13-14

Validation and verification 15 20- Validation and verification 15-20- Validation of Letrozole and Related Substances monograph (USP) 21-27

Monograph methods

Atovaquone oral solution (USP) 28-30Bambuterol (EP) 31-33B d id (EP) 34 36Budesonide (EP) 34-36Dipyridamole (EP) 37-39Dofetilide (USP) 40-42Domperidone (EP) 43-45Fluvoxamine (USP) 46-49Pramipexole (USP) 50-52

From HPLC to UHPLC 53

Fexofenadine – HPLC (USP) 54-56Fexofenadine – UHPLC (USP) 57-58

Olanzapine – HPLC (USP) 59-61p ( )Olanzapine – UHPLC (USP) 62-63

Rizatriptan Benzoate - HPLC (USP) 64-66Rizatriptan Benzoate - UHPLC (USP) 67

Merck Millipore product list 68Merck Millipore product list 68

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Molecular Structures

Atovaquone Bambuterol Budesonide

DipyridamoleDomperidone

Dofetilide

Fexofenadine Fluvoxamine

Letrozole

Ri t i t

3


Olanzapine Pramipexole Rizatriptan

Application IndexMolecule Name Column Used Page

Atovaquone (USP) Chromolith® HighResolution RP 18 endcapped 100x4 6 mm10-11,

Atovaquone (USP) Chromolith® HighResolution RP-18 endcapped, 100x4.6 mm 28-30

Bambuterol (EP) Purospher® STAR RP-18 endcapped (5 μm), Hibar® RT 150x4.6 mm 31-33

Budesonide (EP) Purospher® STAR RP-18 endcapped (3 μm) Hibar® RT 150x4.6 mm 34-37

Dipyridamole (EP) Purospher® STAR RP-18 endcapped (5 μm) Hibar® RT 100x4.6 mm 37-39

Dofetilide (USP) Chromolith® HighResolution RP-18 endcapped, 100x4.6 mm 40-42

Domperidone (EP) Purospher® STAR RP-18 endcapped (3μm) Hibar® RT 100x4.6 mm 43-45

Fexofenadine (USP) – HPLC Purospher® STAR Phenyl (5μm) Hibar® RT 250x4.6 mm 54-56

Fexofenadine (USP) – UHPLC Purospher® STAR Phenyl (2 μm) Hibar® HR 100x2.1 mm 57-58

Fluvoxamine (USP) Purospher® STAR RP-8 endcapped (5μm) Hibar® 250x4.6 mm 46-49

Letrozole (USP) Purospher® STAR RP-18 endcapped (5μm) Hibar® RT 125x4.6 mm 21-27

( ) ( )Olanzapine (USP) – HPLC Purospher® STAR RP-8 endcapped (5μm) Hibar® 250x4.6 mm 59-61

Olanzapine (USP) – UHPLC Purospher® STAR RP-8 endcapped (2μm) Hibar® HR 100x2.1 mm 62-63

Pramipexole (USP) Purospher® STAR RP-18 endcapped (5 μm) Hibar® 150x4.6 mm 50-52

Rizatriptan Benzoate - HPLC Purospher® STAR Phenyl (5μm) Hibar® RT 250x4.6 mm 64-66

Rizatriptan Benzoate - UHPLC Purospher® STAR Phenyl (2μm) Hibar® HR 100x2.1 mm 67

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Introduction

A generic drug (in plural generic drugs or generics) is a drug defined as "a drug product that is /comparable to an ethical drug brand/reference listed drug product; considering dosage, quality

and performance, and intended use. Generics just like ethical drugs must comply with the local regulations of the countries where they are distributed. Thus a generic drug must contain the same active ingredients as the original formulation, within an acceptable bioequivalent range with respect to pharmacokinetic and pharmacodynamic properties.

Generic drugs of biological type are commonly known as biosimilars, and they are different from chemical drugs because of its biological nature and thus regulated under extended set of rules.Biosimilars, or follow-on biologics, are biologic medical products whose active drug substance is made by a living organism. Due to the higher complexity with biological drug manufacturing; small differences in impurities and/or breakdown products can have serious health implications. Thus only few biosimilars have been approved to this date. However, biosimilars is a rather “hot topic” and something currently pursued by numerous companies around the world. In most cases, generic products are available as soon as the patent protection afforded to the originalcases, generic products are available as soon as the patent protection afforded to the original developer have expired. The added market competition with approved generics often lead to substantially lower prices for both the ethical drug product and the generic forms.

In this compilation we have developed a number of HPLC methods for generic small molecule drugs following current monograph methods in the European Pharmacopoeia (EP) and the United States Pharmacopoeia (USP). In particular this compilation presents new data on peak purity analysis as well as an extended discussion about the differences among partial and fullpurity analysis, as well as an extended discussion about the differences among partial and full validation of monograph methods depending if the new method are within allowed changes per the pharmacopoeia. A complete example is shown for method validation within allowed changes (Letrozole and Related Substances), as well as allowed scaling of methods from particulate to monolithic columns. There are also examples of NOT allowed method changes, from HPLC to UHPLC conditions (gradient methods), with substantial time saving in all examples. The benefits with the new methods are obvious and should stimulate you to consider such method transfer d it it ld i l t th d lid tidespite it would require a complete method revalidation.

In all examples, we have used high quality HPLC and UHPLC columns from Merck Millipore combined with our high purity solvents and reagents. Thus examples are given to where the Purospher® STAR (RP-18 endcapped, RP-8 endcapped, and Phenyl stationary phases) as well as Chromolith® HighResolution RP-18 endcapped columns can easily be introduced and used for your needs in validated pharmaceutical quality control.

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Pharmacopoeial Requirements

The regulation of medicinal products date back several hundred years. In the late 15th century King Henry VIII gave the Royal College of Physicians of London the power to inspect apothecaries’ products in the London area and to destroy defective stock. The first list of approved drugs (with manufacturing guidelines) was published in 1618 by the London Pharmacopoeia, and the first edition of the British Pharmacopoeia (BP) was published in 1864 being one of the first attempts to harmonize pharmaceutical standards, through the merger of the London, Edinburgh and Dublin Pharmacopoeias. Today, we can purchase certified reference standards from several official bodies.

The United States Pharmacopoeia (USP) was founded in 1820. Over the last two centuries massive progress has been made around the globe to make drugs safe for consumers, both on a national as well as on an international level, thus assuring the potency of commercially available drug products. Depending where (in which country or region) a finalized drug product will be used it must meet the specified regulations and thus many pharmaceutical companies work from several pharmacopoeias. In this chapter the European (EP) and United States Pharmacopoeiaseveral pharmacopoeias. In this chapter the European (EP) and United States Pharmacopoeia (USP) will be discussed.

One of the more important improvements is the requirement for impurity profiling, i.e. the analysis of related substances (RS) monographs. At present, various regulatory authorities like the International Conference of Harmonization (ICH), European Directorate for the Quality of Medicines and Healthcare (EDQM), United States Food and Drug Administration (USFDA), and the Canadian Drug and Health Agency are all emphasizing the purity requirements and theCanadian Drug and Health Agency are all emphasizing the purity requirements and the identification of impurities in Active Pharmaceutical Ingredients (APIs), and formulated drugs. The ICH Harmonized Tripartite Guideline, ‘Impurities in new drug substances’, Q3A(R2), states that all actual and likely impurities in a new drug substance should be summarized and laboratory studies conducted to detect these impurities. Qualification of the impurities is the process of acquiring and evaluating data that establishes biological safety of an individual impurity; thus revealing the need and scope of impurity profiling of drugs in pharmaceutical

h d f t i Th t id d t th d f h ti l lik lresearch and manufacturing. That said, assay and potency methods of pharmaceuticals are likely the most common chromatographic analysis around the world. The requirements for an assay are less stringent than for an impurity profiling. Contaminants need only be separated from the main component and the focus is more on accuracy, precision, range and linearity.

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Figure 1. The Logotypes of British (BP), European (EP), Indian (IPC), and United States (USP) Pharmacopoeias.

Assay (potency) methods as well as the related substances monographs (identification of impurities) are both carried out with various analytical techniques, and where different chromatographic and spectroscopic techniques are common; either alone or in combination with th t h i Thi l h t h (TLC) hi h f thi l h t hother techniques. Thin layer chromatography (TLC), high performance thin layer chromatography

(HPTLC), gas chromatography (GC), high performance liquid chromatography (HPLC), and atomic absorption spectroscopy (AAS) in addition to more classical tests based on titration are commonly used. HPLC has especially been widely exploited for impurity profiling methods. The reasons for this are the wide range of detectors available that connect easily with HPLC along with the variety of column chemistries (stationary phases) commercially available. Very simply, with HPLC it is possible to develop robust and reliable methods having necessary sensitivity and linearity that meet requirements in selectivity and provide cost effectiveness to the laboratory.

On the following pages, information from USP and EP will be used and explained. A monograph method represents published standards by which the use of one or more substances is automatically authorized. Thus by following the specific method and complying with set specifications a manufacturer can prove the safety of their products. Thus, examples are shown herein how validation of monograph methods can be carried out, and remember, Merck Millipore g p , , panalytical chromatography columns can be an excellent choice for your assay and/or impurity-profiling needs.

Changing a Regulated Method

What changes are allowed in a monograph method?

- Can we change the column material?- Are we allowed to use a different column dimension?- Is it allowed to scale down to smaller ID columns to save solvent?- Is there a possibility to speed up separation?

The answer is “yes” to all these questions...but how?

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Factors that may affect chromatographic behavior:1. Composition, ionic strength, temperature, and apparent pH of the mobile phase

2. Flow rate, column dimensions, column temperature, and pressure

3. Stationary phase characteristics, including type of chromatographic support ( )(particle-based or monolithic), particle or macropore size, porosity, and specific surface area

4. Reversed-phase and other surface modification of the stationary phases, the extent of chemical modification (as expressed by end-capping, carbon loading, etc.)

In some circumstances, it may be desirable to use an HPLC column with different dimensions tothose prescribed in the official procedure (different length, internal diameter, and/or particlesize). In either case, changes in the chemical characteristics (“L” designation) of the stationary phase will be considered a modification to the method and will require full validation.Adjustments to the composition of the mobile phase in gradient elution may cause changes in selectivity and are not recommended. If adjustments are necessary, change in column packing (maintaining the same chemistry), the duration of an initial isocratic hold (when prescribed), and/or dwell volume adjustments are allowed. Additional allowances for gradient adjustment arenoted in the following text and table for USP monographs.noted in the following text and table for USP monographs.

USP EP

Column Length** See separate instructions on next page. NEW in USP 37! ± 70%

Column Inner Diameter See separate instructions on next page. NEW in USP 37! ± 25%Particle Size See separate instructions on next page. NEW in USP 37! Reduction of 50%, no increase

Flow rate See separate instructions on next page. NEW in USP 37! ± 50%

Column Temperature ± 10° C ± 10° C (max 60° C)

Injection Volume can be adjusted as far as it is consistent with acceptedprecision, linearity, and detection limits. Note that excessive injection volume can lead to unacceptable band broadening, causing a reduction in N and resolution. Applies to both gradient and isocratic separations

May be decreased (if LOD and repeatability is OK)

pH ± 0.2 units for both isocratic and gradient separations ± 0.2 unitsUV Wavelength No adjustment is permitted No adjustment is permitted

** A guard column may be used with the following requirements, unless otherwise is indicated in the individual monograph (USP): (a) the length of the guard column must be NMT 15% of the length of the analytical column, (b) the inner diameter must be the same or smaller than that of the analytical column, and (c) the packing material should be the same as the analytical column (e.g., silica) and contain the same bonded phase (e.g., C18). In any case all system suitability requirements specified in the official procedure must be met with the guard column installed

Buffer salts Concentration ± 10% if the permitted pH variation (see above) is met. ± 10%Mobile phase Composition ± 30% relative, or ± 10% absolute whichever is smaller ± 30% relative,

or ± 30% absolute whichever is larger


In any case, all system suitability requirements specified in the official procedure must be met with the guard column installed.

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Changes in USP37

Particle Size (HPLC): For isocratic separations, the particle size and/or the length of the column may be modified provided that the ratio of the column length (L) to the particle size (dp) remains constant or into the range between −25% to +50% of the prescribed L/dp ratio. Alternatively (as for the

)application of particle-size adjustment to superficially porous particles), other combinations of L and dp can be used provided that the number of theoretical plates (N) is within −25% to +50%, relative to the prescribed column.

Caution should be taken when the adjustment results in a higher number of theoretical plates which generates smaller peak volumes, which may require adjustments to minimize extra-column band broadening by factors as instrument plumbing, detector cell volume and sampling rate, and injection volume. When particle size is not mentioned in the monograph, the ratio must be calculated using the largest particle size consigned in the USP definition of the column. For gradient separations, changes in length, column inner diameter and particle size are not allowed.

Flow Rate (HPLC):When the particle size is changed, the flow rate may require adjustment, because smaller-When the particle size is changed, the flow rate may require adjustment, because smallerparticle columns will require higher linear velocities for the same performance (as measured by reduced plate height). Flow rate changes for both a change in column diameter and particle size can be made by:

where F1 and F2 are the flow rates for the original and modified conditions, respectively; dc1 and dc are the respective column diameters; and dp and dp are the particle sizes When a change is

F2 = F1 x [(dc22x dp1)/(dc1

2x dp2)]

dc2 are the respective column diameters; and dp1 and dp2 are the particle sizes. When a change is made from 3 µm to <3 µm particles in isocratic separations, an additional increase in linear velocity (by adjusting flow rate) may be justified, provided that the column efficiency does not drop by more than 20%. Similarly, a change from <3 µm to 3 µm particles may require additional reduction of linear velocity (flow rate) to avoid reduction in column efficiency by more than 20%.

Ch i F d d d t ll d f di t tiChanges in F, dc, and dp are not allowed for gradient separations. Additionally, the flow rate can be adjusted by ±50% (isocratic only). EXAMPLES: Adjustments in column length, internal diameter, particle size, and flow rate can be used in combination to give equivalent conditions (same N), but with differences in pressure and run time. The following table lists some of the more popular column configurations to give equivalent efficiency (N), by adjusting these variables.

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Changes in USP37

Length (L, mm)

Column Diameter (dc, mm)

Particle Size(dp, µm)

Relative Values

L/dp F N Pressure Run Time

250 4.6 10 25000 0.5 0.8 0.2 3.3

150 4.6 5 30000 1.0 1.0 1.0 1.0

150 2.1 5 30000 0.2 1.0 1.0 1.0

100 4.6 3.5 28600 1.4 1.0 1.9 0.5

100 2 1 3 5 28600 0 3 1 0 1 9 0 5100 2.1 3.5 28600 0.3 1.0 1.9 0.5

75 4.6 2.5 30000 2.0 1.0 4.0 0.3

75 2.1 2.5 30000 0.4 1.0 4.0 0.3

50 4.6 1.7 29400 2.9 1.0 8.5 0.1

For example, if a monograph specifies a 150×4.6 mm; 5 µm column operated at 1.5 mL/min, the same separation may be expected with a 75×2.1mm; 2.5 µm column operated at 1.5 mL/min ×0 4 = 0 6 mL/min along with a pressure increase of about four times and a reduction in run time

50 2.1 1.7 29400 0.6 1.0 8.5 0.1

0.4 = 0.6 mL/min, along with a pressure increase of about four times and a reduction in run time to about 30% of the original.

Injection Volume (HPLC): The injection volume can be adjusted as far as it is consistent with accepted precision, linearity,and detection limits. Note that excessive injection volume can lead to unacceptable band broadening, causing a reduction in N and resolution. Applies to both gradient and isocratic

tiseparations.

The easiest approach to scale the injection volume is to compare differences in column tube volume and to keep same volumetric ratio between tube volume and injection volume, and thereby same volume loading on the column. A method scaled from a 250x4.6 to 100x2.1 mm column require a 12-fold reduction of injection volume using simple volume calculation of a tube (i.e. 250x4.6 =4.15 mL and 100x2.1 =0.346 mL). Thus if injection volume is 20 µL on the


larger column, it is recommended to inject not more than 2 (1.7) µL on the smaller column.

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Ratio of Components in Mobile PhaseThe following adjustment limits apply to minor components of the mobile phase (specified at 50% or less). The amounts of these components can be adjusted by ±30% relative. However, the change in any component cannot exceed ±10% absolute (i.e., in relation to the total mobile phase). Adjustment can be made to one minor component in a ternary mixture. Examples of adjustments for binary and ternary mixtures are given below.

Binary Mixtures specified ratio of 50:50. 30% of 50 is 15% absolute, but this exceeds the maximum permitted change of ±10% absolute in either component. Therefore, the mobile phase ratio may be adjusted only within the range of 40:60 to 60:40 specified ratio of 2:98: 30% of 2 is 0.6% absolute. Therefore the maximum allowed adjustment is within the range of 1.4:98.6 to 2.6:97.4.

Ternary Mixtures specified ratio of 60:35:5. For the second component, 30% of 35 are 10.5% absolute, which exceeds the maximum permitted change of ±10% absolute in any component. Therefore the second component may be adjusted only within the range of 25% to 45% absolute. For the third component, 30% of 5 is 1.5% absolute. In all cases, a sufficient quantity of the first component is used to give a total of 100%. Therefore, mixture ranges of 50:45:5 to 70:25:5 or 58.5:35:6.5 to 61.5:35:3.5 would meet the requirement.

Wavelength of UV-Visible DetectorDeviation is not permitted from the specified wavelength. The procedure specified by the detector manufacturer, or another validated procedure, is used to verify that error in the detector wavelength is, at most, ±3 nm.

Choosing the right Column to meet Monograph SpecificationsThe HPLC column choice is a very important consideration or it will be difficult to meet the setThe HPLC column choice is a very important consideration or it will be difficult to meet the set requirements in a monograph method. In the chapter discussing column selection, we have outlined which USP classification (code) our HPLC columns belong to. At present, Merck Millipore offers L1, L3, L7, L8, L10, L11, L20, L29 and L45 modifications.

In addition, the USP has a database for chromatography columns to help users cross-reference HPLC columns. However, it is important to keep in mind that this database is only a tool as “the d t b it lf i t t f th t t f USP NF d d t tit t ffi i l i t t ti fdatabase itself is not part of the text of USP–NF, and does not constitute an official interpretation of such text. The databases being displayed at the site are provided for informational purposes only to assist users in finding HPLC columns equivalent to that used to develop and validate a particular chromatographic procedure. After finding suggestions of equivalent columns using the databases, the columns should be tested with the appropriate sample. USP and the authors of the databases are not responsible for the results obtained with the columns proposed by the databases and such results should not be relied on to demonstrate compliance with USP standards or requirements. The data being provided by the databases were generated using brand new columns USP has no information on and disclaims any


by the databases were generated using brand new columns. USP has no information on and disclaims any knowledge of how these procedures will perform when evaluating already used columns”.

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USP Packings (L classifications)

Packing Description Chemistry

L1 Octadecylsilane chemically bonded to porous silica or RP 18 (C or ODS)L1 Octadecylsilane chemically bonded to porous silica or ceramic micro-particles, 1.5 to 10 μm in diameter, or a monolithic rod.

RP-18 (C18 or ODS)

L3 Porous silica particles, 1.5 - 10 μm in diameter, or a monolithic rod.

Silica (Si)

L7 Octylsilane chemically bonded to totally porous or RP-8 (C8)superficially porous silica particles 1.5 to 10 μm in diameter, or a monolithic rod.

L9 An essentially monomolecular layer of aminopropylsilane chemically bonded to totally porous silica gel support, 1.5 to 10 μm in diameter.

NH2

L10 Nitrile groups chemically bonded to porous silica CNg p pparticles 1.5 to 10 μm in diameter

L11 Phenyl groups chemically bonded to porous silica particles 1.5 to 10 μm in diameter.

Phenyl

L20 Dihydropropane groups chemically bonded to porous silica particles, 1.5 to 10 μm in diameter.

Diol

L G l i h l b Al iL29 Gamma alumina, reverse-phase, low carbon percentage by weight, alumina-based polybutadiene spherical particles, 5 μm in diameter with a pore volume of 80 angstrom units.

Alumina

L45 Beta cyclodextrin, R,S-hydroxypropyl ether derivative, bonded to porous silica particles, 5 - 10 μm in diameter.

Cyclodextrin(Chiral)

A discussion with USP was initiated early in 2014, to further extend the inclusion of...or a monolithic rod to the L9, L10, L11 and L20 packings definition. This update is not yet in practice.


We at Merck Millipore have shown that our columns can easily meet monograph specifications despite the fact that they may seem very different from the column used when developing the original monograph method. Also it important to keep in mind that those columns mentioned in USP as monograph columns is not bound text – the actual monograph only describe the column geometry and classification.

System Suitability Test (SST)To verify and validate a monograph method and meet set requirements defined, system suitability tests are described.

1. SST is used to verify that the chromatographic system is adequate for the intended analysis.2. SST is based on the concept that the equipment, electronics, analytical operations, and

samples analyzed constitute an integral system that can be evaluated as such

As long as the changes of a monograph method are within the limits shown above it is possible to carry out only a partial revalidation followed by internal documentation of the updated method. If the changes are beyond limits, a complete revalidation and documentation is required followed by a discussion with an auditor and regulating authorities for approval of the new method. It is (of course) also possible to submit completely new monograph methods to ( ) p p y g pauthorities.

Atovaquone Oral Suspension (USP37–NF32) – Case StudyThe current monograph for Atovaquone oral suspension is based on HPLC where the liquid chromatograph is equipped with a UV detector (220 nm) and a 125x4.6 mm column that contains packing L1 (RP-18/ODS) operated at 3.0 mL/min. No specific particle size is mentioned in the monographin the monograph.

At this flow rate, with normal particulate columns (5 μm), the back pressure over the HPLC system will be very high on a fresh column such that after a few runs, it will exceed the set backpressure limit and causes failure in analysis.

For these reasons we have transferred the current method to a 100x4.6 mm id monolithic l Ch lith® Hi hR l ti RP 18 d d l Fi 2 h th b kcolumn, Chromolith® HighResolution RP-18 endcapped column, see Figure 2, where the back

pressure is only around 100 bar (1440 psi) under given experimental conditions. A pressure that any HPLC system easily can accommodate, and thereby provide robust and reliable results over time.


20.4

20.5

Atovaquone RS A

20.1

20.2

20.3

Inte

nsit

y (m

V)

Sample solution

Fi 2 Ch t hi lt f th t l l ti (0 09 / L t f th l

200 2 4 6 8 10

Retention time (min)

Figure 2. Chromatographic results of the atovaquone sample solution (0.09 mg/mL atovaquone from the sample stock solution) at 3.0 mL/min flow rate on a 100x4.6 mm id Chromolith® HighResolution RP-18 endcapped column. The system backpressure under these conditions is only 104 bar (1510 psi).

Chromatographic Data:No. Compound Retention Time (min) RRT Tailing factor Theoretical Plate

As illustrated by Figure 2 and the performance table above, both the criteria for peak tailing (NMT 1 5) and the relative retention time (RRT) for Atovaquone related compound A and

1 Atovaquone related compound A 3.93 0.88 - -

2 Atovaquone 4.43 1.00 1.2 9466

(NMT 1.5), and the relative retention time (RRT) for Atovaquone related compound A and Atovaquone is met. Further details of this method can be found on page 28-30.

The method transfer is within allowed changes for partial revalidation.


United States PharmacopoeiaThe process of validating a new analytical procedure for compendial usage is addressed in USP general Chapter 1225 – “Validation of Compendial Procedures”. However, even with a fully validated procedure, the end-user may not have assurance that the procedure is suitable for use with a specific ingredient or product in a specific laboratory with specific personnel, equipment, consumables and reagents. USP therefore developed chapter 1226 in response to industry’s request to provide instructions for verifying compendial procedures in specific situations. Here we have addressed USP’s proposed new general chapter 1226 ”Verification of Compendial Procedures” which is intended to fill the gap in the proper usage of compendial procedures by outlining a process for verifying their suitability. The role of HPLC columns is of immense importance to meet system suitability test (SST) criteria in compendial methods.

Validation of Compendial Procedure <1225> p

1. Defines analytical performance characteristics2. Recommends data for submission to USP-NF3. Provides guidance on which analytical performance characteristics are needed based on the

type of test4. Incorporates ICH guidelines Q2A and Q2B

Performance Characteristics

Category 1 Category 2 Category 3 Category 4

Quantitative Limit Test

Accuracy Yes Yes - - NoPrecision Yes Yes No Yes No

Specificity Yes Yes Yes - Yes

Verification of Compendial Procedures <1226>

The intention of this USP chapter is to provide general information to laboratories on the

LOD No Yes Yes - NoLOQ No No No - NoLinearity Yes Yes No - NoRange Yes Yes - - No

verification of compendial procedures that are being performed for the first time to yield acceptable results utilizing the laboratories’ personnel, equipment, and reagents.

Verification consists of assessing selected Analytical Performance Characteristics, such as those described in chapter 1225, to generate appropriate, relevant data rather than repeating the validation process. The table below illustrates required tests for the USP chapters dealing with validation and verification.


Performance Validation Verification

Accuracy Yes No

Precision Yes Maybe

Specificity Yes Yes

LOD No No

LOQ Yes Yes

Why USP <1226> is needed:

1. 21 CFR211.194 (a)(2): “users of analytical methods described in USP–NF are not required to validate the accuracy and reliability of these methods, but merely verify their suitability under actual conditions of use”.R i d i i i

Linearity Yes No

Range Yes No

2. Response to industry inquiries3. Verification consist of assessing selected Analytical Performance Characteristics, such as

those which are described in USP Chapter 1225, to generate appropriate, relevant data rather than repeating the validation process.

Reference Standards“Reference Standards provided by the United States Pharmacopeial Convention (USP Reference Standards, or RS) are highly characterized specimens reflective of specified drugs and foods (drug substances, biologics, excipients, dietary supplements, food ingredients, impurities, degradation products, reagents, and performance verification standards). When approved as suitable for use as comparison standards for documentary tests or assays (i.e., as a monograph component) in the United States Pharmacopeia (USP) or National Formulary (NF), USP RS also assume official status and legal recognition in the United States. Assessment of the suitability for use in other applications rests with the user. Official USP RS are primary standards infor use in other applications rests with the user. Official USP RS are primary standards in jurisdictions that so recognize them as such and, when appropriate, are calibrated relative to international reference materials such as those provided by the World Health Organization.”

USP Monograph ModernizationThe United States Pharmacopeia (USP) develops and publishes monographs and general chapters that provide public quality standards for drugs, excipients, and dietary supplements in the United St t Ph i d th N ti l F l (USP NF) USP h t t d l b l i iti ti tStates Pharmacopeia and the National Formulary (USP–NF). USP has started a global initiative to modernize many of the existing monographs and is actively seeking industry collaborators to assist in the development of such monographs. The direct participation of the pharmaceutical industry, and other interested stakeholders in this program are encouraged to assist in providing updated public standards to strengthen the protection of public health. USP intends to modernize these monographs as soon as possible; either by traditional submission from a stakeholder or from USPs internal laboratory efforts. For more information, please contact the


Standards Acquisition Department at [email protected].

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Method Validation (Assay method) The following pages introduces a procedure how to plan method validation per pharmacopoeial requirements.

Specificity test:1 U bl k l ti t h i t f1. Use a blank solution to show no interference 2. Use placebo to demonstrate the lack of interference from excipients 3. Use spiked samples to show that all known related substances are resolved from each other4. Use stressed samples with about 10 to 20% degradation are used to demonstrate the

resolution among degradation products. Check the peak purity of the drug substance by using a photodiode array detector (PDA); e.g. purity angle is lower than the purity threshold.

5. Representative chromatograms should be provided with time scale and attenuation indicated

Linearity and Range:From the calibration curve we can calculate the slope and from the slope we can calculate the limit of detection (LOD) and limit of quantitation (LOQ). In this example, the 100% concentration level corresponds to 75 ppm of main molecule, see table below.

Concentration (ppm) % to concentration112.5 150

LOQ Repeatability:

90 12075 10060 80

37.5 5018.75 257.5 103.75 50.75 1

LOQ Repeatability:Good repeatability at LOQ level is a requirement and average value, the deviation from mean and the relative standard deviation in % should be calculated from minimum 10 injections, see table.

Injection Number Area12345678910

Average

Standard Deviation (SD)Relative Standard deviation (RSD) %


Relative Standard deviation (RSD) %

Accuracy and Recovery:At least three different standard concentrations should be analyzed from final method sample concentrations; in this example and as in most cases for the LOQ level, 25% and 50 % level. A sample set can be designed in the following way (using standard addition technique):

Sample Concentration (ppm) Recovery level Area

75 0 (means no addition to standard concentration 75 ppm)( pp )

75 LOQ (means addition of LOQ concentration + 75 ppm)

75 25 % (means addition of 25% level = 18.75 + 75 ppm)

75 50 % (means addition of 50% level = 37.5 + 75 ppm)LOQ

(From calibration curve under linearity)25% (means 18.75)50% (means 37.5)

Recovery or accuracy can be calculated by the difference in area of pure sample and recovery standards and should be reported in percentage (%). The acceptable limit is 85-110 %.

Precision: To determine the method precision multiple measurements of a defined sample should be carried out by the same analyst. A minimum of six (6) injections at the test concentration (6 times of a single batch) must be carried out, or to perform analysis at three (3) different concentration levels

( )

single batch) must be carried out, or to perform analysis at three (3) different concentration levels (80, 100, and120%) with three (3) repetitions each. The latter approach is a convenient way to also allow for method accuracy determination. For a monograph assay method the relative standard deviation should be better than 2.0% (RSD < 2.0%).

Intermediate Precision:Ruggedness should be determined by analysis of same sample on multiple days, with multiple analysts and multiple equipments Repeat the method precision by different analyst in differentanalysts, and multiple equipments. Repeat the method precision by different analyst in different equipment using different column lots on different days (three different column lots are generally recommended). The RSD should be within same level as for the method precision. For a monograph assay method the relative standard deviation should be better than 2.0% (RSD < 2.0%).

Robustness:T d t i th th d' bilit t i ff t d ll b t d lib t i ti iTo determine the method's capability to remain unaffected, small but deliberate variations in method parameters are carried out and where the following changes can be used as a recommendation. 1. Influence of variations of pH in a mobile phase (Changing buffer pH by +/- 0.2 units)2. Influence of variations in mobile phase composition (Change in organic composition +/- 2.0%)3. Test different columns (different lots and/or suppliers)4. Temperature


5. Flow rate (+/- 0.2˚C)

18

The acceptance criteria are that the system suitability test (SST) parameters should pass for all the conditions and all known and unknown impurities shall be well separated from each other.

Validation Protocols (Related Substances) A related substances method commonly have more stringent requirements, specifying relative retention time (RRT) and some time the relative response factors (RRF). From RRT, the retention time of the impurity can be identified and from RRF one may calculate the percentage of impurities present in the sample. As an example, let us look at the current Clarithromycin related substances method (USP37-NF32). The RRT and RRF given for Clarithromycin and 16 Clarithromycin impurities are given in the following table.

Name RRT RRF

Clarithromycin impurity I 0.38 1.0Clarithromycin impurity A ( Clarithromycin F) 0.42 1.0Clarithromycin impurity J 0.63 1.0Clarithromycin impurity L 0.74 1.0Clarithromycin impurity B 0.79 1.0Clarithromycin impurity M 0.81 1.0Clarithromycin impurity C 0.89 1.0Clarithromycin impurity D 0.96 1.0Cl ith i 1 0Clarithromycin 1.0 -Clarithromycin impurity N 1.15 1.0Clarithromycin related compound A 1.27 1.0Clarithromycin impurity F 1.33 1.0Clarithromycin impurity P 1.35 1.0Clarithromycin impurity O 1.38 1.0Clarithromycin impurity K 1.59 1.0Clarithromycin impurity G 1.72 3.7Clarithromycin impurity H 1.82 6.7

The percentage of each impurity is calculated using the following formula:

Result = (rU/rS)×(CS/CU)×(1/F )×100rU = peak response of any individual impurity (related substances; RS) from the Sample solutionrS = peak response of Clarithromycin from Standard solution 3CS = concentration of USP Clarithromycin RS in Standard solution 3 (mg/mL)CU = concentration of Clarithromycin in the Sample solution (mg/mL)F = Relative response factor as mentioned in table

The validation of Clarithromycin related substances can be carried out using Standard Solution 3 (specific details are given in the current monograph but not important for the general discussion here.), setting the Clarithromycin concentration as 100% concentration and following the same procedure as described in the assay. The concentration of any individual impurity should not be


p y y p ymore than 0.4 % and total impurities NMT 3.5%.

19

Using then Standard solution 1 (Clarithromycin concentration is 1.5 mg/m L or 1500 ppm, the 0.4 % level (of 1500 ppm) correspond to 6 ppm. If any individual impurity standard is available then validation should be carried out using a 6 ppm concentration as 100% and repeat analysis as described in the assay.

Linearity and Range (see Assay procedure):Linearity and Range (see Assay procedure):From the calibration curve we can calculate the slope and from the slope we can calculate the limit of detection (LOD) and limit of quantitation (LOQ). In this example, the 100% concentration level corresponds to 6 ppm of USP Clarithromycin RS.

LOQ Repeatability, Accuracy and Recovery:For RS monograph method it possible to proceed as described in the assay method. The difference is that the relative standard deviation must not be more than 10% for the relateddifference is that the relative standard deviation must not be more than 10% for the related substances, and the concentration of Clarithromycin RS sample solutions are much lower, i.e. 6 ppm, LOQ, 25% and 50%.

Injection Number Area

12345678910

Average

Standard Deviation (SD)Standard Deviation (SD)Relative Standard deviation (RSD) %

Sample Concentration (ppm) Recovery level Area

6.0 0 (means no addition to standard concentration 6 ppm)

6.0 LOQ (means addition of LOQ concentration + 6 ppm)

6.0 25 % (means addition of 25% level = 1.5 + 6 ppm)

On the following pages, a complete method validation is presented for the Letrozole and Related Substances monograph method (USP)

( pp )

6.0 50 % (means addition of 50% level = 37.5 + 6 ppm)LOQ

(From calibration curve under linearity)25% (means 1.5)50% (means 3.0)

Letrozole and Related Substances monograph method (USP).


Letrozole and Related Substances (USP)Purospher® STAR RP-18 endcapped

Letrozole

Letrozole is an oral non-steroidal aromatase inhibitor for the treatment of hormonally-responsive breast cancer after surgery. Estrogens are produced by the conversion of androgens through the activity of the aromatase enzyme. Estrogens then bind to an estrogen receptor, which causes cells to divide. Letrozole prevents the aromatase from producing estrogens by competitive, reversible binding to the heme of its cytochrome P450 unit. The action is specific, and Letrozole does not reduce production of mineralo- or y p , pcorticosteroids. The commercial trade name of Letrozole is Femara.

The current USP monograph method for Letrozole and related substances specifies use of a 125x4.6 mm column with L1 (RP-18/ODS) (5 μm) packing as stationary phase, and with identical experimental conditions as described in the assay method. System suitability requirements for related substances are provided by means of resolution between Letrozole and Letrozole related substance A and should not be less than 2.0, as well as a relative retention time RRT of 0.67 Letrozole related substance A, 1.0 for Letrozole and 2.4 for 4,4',4''-Methanetriyltribenzonitrile. The tailing factor should be 0.8-1.5 for Letrozole.

The following pages illustrate a complete procedure for the method validation within limits of allowed changes. All acceptance critera are met for the Letrozole and related substances method by using the identical matched column; 125x4.6 mm Purospher® STAR RP-18 endcapped (5 μm) column for Letrozole analysis.


Chromatographic ConditionsCol mn P h ® STAR RP 18 d d (5 ) Hib ® RT 125 4 6 1 51914 0001


Column: Purospher® STAR RP-18 endcapped (5μm) Hibar® RT 125x4.6 1.51914.0001Injection: 20 μLDetection: UV 230 nmCell: 10 μLFlow Rate: 1.0 mL/minMobile Phase: A: Water

B: AcetonitrileGradient: See table

Time (min)

A(%)

B(%)

0.01 70 30

25.0 30 70

26.0 70 20Gradient: See tableTemperature: 25˚CDiluent: Acetonitrile and water; 3:7 (v/v)SST solution: 2 μg/mL of Letrozole Related Compound A and 10 μg/mL of Letrozole in DiluentStandard solution: 1 μg/mL of USP Letrozole in Diluent.Sample solution: Transfer 25 mg of Letrozole to a 250-mL volumetric flask.

Dissolve in 75 mL of acetonitrile, and dilute with water to volume.Pressure Drop: 72 - 28 Bar (1044 - 410 psi)

26.0 70 20

30.0 70 30

240

320

400

ty (m

V)

Blank

240

320

400

ty (m

V)

SST solution

zole

RS A

0

80

160

0 5 10 15 20 25 30

Inte

nsit


0

80

160

0 5 10 15 20 25 30

Inte

nsit


Letr

o

Chromatographic Data :

No.

Compound Retention Time (min)

RRT Resolution Theoretical Plate Assymetry

1 Letrozole related compound A 4.9 0.62 11641 1.10

2 Letrozole 7 9 1 0 14 3 20133 1 072 Letrozole 7.9 1.0 14.3 20133 1.07



Peak Purity curve: Letrozole RS A

0.8

0.9

1.0

32.5

35.0

37.5

40.0

mAUPeak

Zero LinePurity Curve

UV spectra: Letrozole RS A

65

70

75

80

85

90

mAU 4.81/ 1.00

20

0

0.2

0.3

0.4

0.5

0.6

0.7

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0

10

15

20

25

30

35

40

45

50

55

60

21

5

24

1

4.65 4.70 4.75 4.80 4.85 4.90 4.95 5.00 5.05 min

0.0

0.1

0.0

2.5

5.0

5

200.0 225.0 250.0 275.0 300.0 325.0 350.0 nm

-5

0

5

26

4

27

3

Peak Purity curve: Letrozole UV spectra: Letrozole

0 45

0.50

0.55

0.60

0.65

0.70

0.75

0.80

0.85

0.90

0.95

80

90

100

110

120

130

140

150

mAUPeak

Zero LinePurity Curve

90

100

110

120

130

140

150

160

170

180

190

200mAU

7.66/ 1.00

200

240

7.4 7.5 7.6 7.7 7.8 7.9 8.0 min-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0

10

20

30

40

50

60

70

200.0 225.0 250.0 275.0 300.0 325.0 350.0 nm

-10

0

10

20

30

40

50

60

70

80

215

262

29627

3

325


M th d V lid ti


Method Validation

Following parameters have been checked during the validation of Letrozole

Specificity Precision and Robustness Linearity

Specificity : Each individual component of the sample has been analysed using the same conditions. Not a single component or blank has any interference on each other. The method stands specific using the Purospher® STAR RP 18 endcapped column

Linearity Limit of Detection (LOD) and Limit of Quantitation (LOQ) LOQ repeatability

The method stands specific using the Purospher® STAR RP-18 endcapped column.

300000

350000

400000

uV

100000

150000

200000

250000

Letrozole Related compound A

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min

-100000

-50000

0

50000


Precision:


The retention time precision achieved for both cases is < 0.5 % RSD. The area precision achieved for both the cases is < 1 % RSD.

800000

900000

1000000uV

Table I: System Precision

Retention Time (min) Area

Injection RS A Letrozole RS A Letrozole

1 4 98 7 90 474836 2457649

100000

200000

300000

400000

500000

600000

700000

1 4.98 7.90 474836 2457649

2 4.97 7.89 474364 2458516

3 4.97 7.89 474541 2450935

4 4.96 7.87 474747 2449480

5 4.95 7.86 474431 2450486

6 4.93 7.84 473816 2451989

A

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0min

-100000

0

100000

Table II: Method Precision

Average 4.96 7.874 474456 2453176

Std. Dev. 0.02 0.023 362 3895

% RSD 0.40 0.29 0.08 0.16

Retention Time (min) Area900000

uV

Injection RS A Letrozole RS A Letrozole

1 4.93 7.85 478983 2473247

2 4.95 7.87 479259 2478348

3 4.95 7.87 480187 2479408

4 4.94 7.86 479478 2479989

5 4 94 7 86 480206 2477724

400000

500000

600000

700000

800000

900000

5 4.94 7.86 480206 2477724

6 4.95 7.87 478293 2486492

Average 4.94 7.863 480206 2479201

Std. Dev. <0.01 <0.01 3294 4294

% RSD 0.12 0.09 0.69 0.17

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0min

-100000

0

100000

200000

300000


Linearity:


2.25

2.50

2.75

3.00

Conc.

11

12

13

14

15

Conc.

Letrozole Related Substance A Letrozole

y = 240765x - 1154.2R² = 0.9998

y = 250992x - 10622R² = 0.9999

0.50

0.75

1.00

1.25

1.50

1.75

2.00

2

3

4

5

6

7

8

9

10

0 50000 100000 150000 200000 250000 300000 350000 400000 450000 500000 550000 600000 650000 700000 Area0.00

0.25

0 500000 1000000 1500000 2000000 2500000 3000000 3500000 Area0

1

550000

600000

650000

uVLetrozole RS A Letrozole

Conc(ppm)

Area Conc(ppm)

Area

200000

250000

300000

350000

400000

450000

500000 0.02 4394 0.1 22557

0.1 22630 0.5 116715

0.2 43882 1.0 229577

0.5 120464 2.5 631260

1.0 240666 5.0 1231279

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0min

0

50000

100000

150000 1.6 380178 8.0 1976461

2.0 487146 10.0 2519018

2.4 578138 12.0 3001958

3.0 717196 15.0 3722293

LOD for Related Compound A 0.03 ppmLOQ for Related Compound A 0 1 ppmLOQ for Related Compound A 0.1 ppm


LOQ repeatability Related Substance A:


LOQ repeatability Related Substance A:

70000

80000

90000

uV

Injection Area

1 22718

2 22826

3 22825

30000

40000

50000

60000

3 22825

4 22730

5 22799

6 22498

7 22622

8 22346

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0min

-10000

0

10000

200008 22346

9 22291

10 22555

Average 22621

Std. Dev 194.54

% RSD 0 86 %0.86 %

Relative standard deviation (RSD in %) for Letrozole Related Substance A at LOQ level 0.86%

Conclusion:Conclusion:

The analytical HPLC method for Letrozole related substance has been validated and it passed all method validation criteria with the Purospher® STAR RP-18 endcapped column.


Atovaquone Oral Suspension (USP)

Atovaquone belongs to the class of naphthoquinones; a hydroxy-1,4-naphthoquinone, an analog of ubiquinone, with antipneumocystic activity. It is manufactured in the US in the liquid form, or oral suspension, under the brand name Mepron.

The current USP monograph for Atovaquone oral suspension specifies the use of a L1 (RP-18) column with 125x4.6 mm geometry (no particle size mentioned), used at a flow rate of 3.0 mL/min.

At this flow rate, with normal particulate columns (5 μm), the back pressure over the HPLC system will be very high on a fresh column, and only after some analyses it goes beyond the sustainable backpressure limit and causes failure in analysis.

Of these reasons we have transferred the current method to a 100x4.6 mm id monolithic column, i.e. a Chromolith® HighResolution RP-18 endcapped column, where the back pressure is only around 100 bar (1440 psi) under given experimental conditions. A pressure that any HPLC system easily can accommodate, and thereby provide robust and reliable results over time.

The method meet all acceptance criteria with good reproducibility for the specified sample solution per current monograph, and will not require a complete re-validation for acceptance.


Chromatographic ConditionsColumn: Chromolith® HighResolution RP-18 endcapped, 100x4.6 mm 1.52022.0001

Atovaquone Oral Suspension (USP)Chromolith® HighResolution RP-18 endcapped

Column: Chromolith HighResolution RP 18 endcapped, 100x4.6 mm 1.52022.0001Injection: 20 μLDetection: UV 220 nmCell: 10 μLFlow Rate: 3.0 mL/minMobile Phase: Acetonitrile, methanol, water, and phosphoric acid; 480:160:360:5 (v/v/v/v)Temperature: 30˚CDiluent: 0.1 M methanolic sodium hydroxideSST Solution: Nominally 3 mg/mL from a known volume of well-mixed oral suspension y g/ pStandard Solution: Not less than (NLT) 750 mg of atovaquone prepared as following procedure:

In an appropriately sized, low-actinic volumetric flask, add 20% volume of water, swirl for 5 min, add 60% volume of 0.1 M methanolic sodium hydroxide, and sonicate for 15 min. Allow to cool, and dilute with 0.1 M methanolic sodium hydroxide to volume. Immediately filter a 20-ml portion, discarding the first 5 mL of the filtrate.

Sample solution: 0.09 mg/mL of atovaquone from the clear filtrate of the Sample stock solution. Transfer to an appropriately sized, low-actinic volumetric flask, and dilute with a mixture of methanol and water (1:1) to volume Minimize exposure of this solution to lightmethanol and water (1:1) to volume. Minimize exposure of this solution to light.

Pressure Drop: 104 Bar (1510 psi)

50100150200250300

nten

sity

(mV)

Sample solution

050

0 2 4 6 8 10

In


150200250300

sity

(mV)

Blank

050

100

0 2 4 6 8 10

Inte

n

Retention time (min)Chromatographic Data :No. Compound Retention Time (min) Tailing factor Theoretical Plate

Suitability requirement:NMT 1.5

1 Atovaquone 4.4 1.2 9466


Atovaquone Oral Suspension (USP)Chromolith® HighResolution RP-18 endcappedRepeatability Data

The relative standard deviation (RSD) for six repeated analyses of the sample solution (0.09 mg/mL of atovaquone) were excellent with far better precission than the specified suitability requirement (NMT 2.0%).

InjectionNumber

Retention time

Area900000

Number time

1 4.42 1786281

2 4.43 1784126

3 4.43 1784598

4 4.42 1786092

5 4 42 1785619400000

500000

600000

700000

800000

5 4.42 1785619

6 4.43 1788002

Average 4.43 1785786

Standard Deviation 0.00 1374

RSD (%) 0.04 0.08

0

100000

200000

300000

Recommended chemicals and reagents to reproduce this alternative method

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

Recommended chemicals and reagents to reproduce this alternative method

Product Name of the Chemical Article Number

1 ortho-Phosphoric acid 85% for analysis EMSURE® ACS, ISO, Reag. Ph Eur 1.00573

2 Sodium hydroxide pellets for analysis (max. 0.02% K) EMSURE® ACS, Reag. Ph Eur 1.06469

3 Acetonitrile gradient grade for liquid chromatography LiChrosolv® Reag. Ph Eur 1.00030

4 Methanol gradient grade for liquid chromatography LiChrosolv® Reag. Ph Eur 1.06007


Bambuterol and Related Substances (EP)

Bambuterol Formoterol

Bambuterol is a long acting beta-adrenoceptor agonist (LABA) used in the treatment of asthma. It is also a prodrug of terbutaline. Commercially, bambuterol is marketed as Bambec and Oxeol.

Formoterol or eformoterol is a long-acting β2 agonist used in the management of asthma and chronicFormoterol or eformoterol is a long acting β2 agonist used in the management of asthma and chronic obstructive pulmonary disease (COPD). It is marketed in four forms: a dry-powder inhaler, a metered-dose inhaler, an oral tablet, and an inhalation solution, under various trade names including Foradil/Foradile, Oxeze/Oxis, and (with Budenoside) Symbicort, Atock, Atimos, and Perforomist.

The current EP monograph for Bambuterol and related substances specifies the use of a 150x4.6 mm column with base-deactivated octadecylsilyl (ODS or RP-18) silica gel for chromatography R (5 μm). The total run-time must be 1.5 times the retention time of bambuterol, with the following retention times; formoterol = about 7 min; bambuterol = about 9 min. If necessary, the composition of the mobile phase can be adjusted; increase the content of phosphate buffer to increase the retention time to meet a chromatographic resolution of minimum 5.0 between the bambuterol and formoterol peaks.

The following pages illustrate that acceptance critera are being met, and that a Purospher® STAR RP-18 endcapped (5 μm), Hibar® RT 150x4.6 mm column is an excellent alternative for Bambuterol analysis.


Bambuterol and Related Substances (EP)Purospher® STAR RP-18 endcappedChromatographic ConditionsColumn: Purospher® STAR RP 18 endcapped (5 μm) Hibar® RT 150x4 6 mm 1 51455 0001Column: Purospher® STAR RP-18 endcapped (5 μm), Hibar® RT 150x4.6 mm 1.51455.0001Injection: 20 μLDetection: UV, 214 nmCell: 10 μLFlow Rate: 1.5 mL/minMobile Phase: Dissolve 6.90 g of sodium di-hydrogen phosphate monohydrate in water and dilute to 1000 mL

with water. Adjust to pH 3.0 with a 50 g/L solution of dilute phosphoric acid. Dissolve 1.3 g of sodium octanesulfonate in 430 mL of a mixture of 25 volumes of acetonitrile and 75 volumes of methanol and 570 ml of buffer solution75 volumes of methanol and 570 ml of buffer solution.

Temperature: AmbientDiluent: Mobile phaseTest Solution: 5.0 mg Bambuterol in 10 ml mobile phase.

Resolution Solution:

Dissolve 1.0 mg of formoterol fumarate dihydrate in the mobile phase and dilute to 10.0 mL with the mobile phase. Mix 0.8 mL of this solution with 0.4 mL of the test solution and dilute to 100.0 mL with the mobile phase.

Pressure Drop: 224 Bar (3248 psi)

2

3

4

5

tens

ity

(mV)

Test solution

Formoterol

4

5

V) Blank

0

1

0 5 10 15 20 25 30

In


0

1

2

3

0 5 10 15 20 25 30

Inte

nsit

y (m

V Blank



Bambuterol and Related Substances (EP)Purospher® STAR RP-18 endcapped

5

3

4

5

ensi

ty (m

V)

Resolution solution

0

1

2

0 5 10 15 20 25 30

Int Resolution solution


Chromatographic Data:No. Compound Retention Time (min) Resolution RRT

1 Formoterol 9.7 0.75

2 B b t l 12 9 1 00

Suitability requirement: NLT 5.0

2 Bambuterol 12.9 6.2 1.00

Peak purity curve - Bambuterol

0.75

1.00

500

600

mAU

PeakZero Line

UV spectra: Bambuterol

750

mAU

12.90/ 1.00

19

3

12.5 13.0 13.5 14.0 min

0.00

0.25

0.50

0

100

200

300

400

200 250 300 350 nm

0

250

500

23

8

31

5

33

1

19

9

26

4

35

0


Budesonide and Related Substances (EP)

Budesonide

Budesonide is a glucocorticoid steroid for the treatment of asthma, COPD and non-infectious rhinitis (including hay fever and other allergies), and for treatment and prevention of nasal polyposis. In addition, it is used for Crohn's disease (inflammatory bowel disease).

A new extended-release formulation of budesonide called "Uceris" has been recently approved (2013) by the United States FDA for ulcerative colitis.

The current EP monograph method for Budesonide and related substances specifies the use of a 150x4.6 mm column with and end-capped octadecylsilyl (ODS or RP-18) silica gel for chromatography R (3 μm). The relative retention with reference to budesonide epimer B (retention time = about 17 min);impurity A = about 0.1; epimers of impurity D = about 0.63 and 0.67; impurity L = about 0.95; epimers ofimpurity G = about 1.2 and 1.3; epimers of impurity K = about 2.9 and 3.0. The system suitability criteria also specify a minimum resolution of 1.5 between the 2 principal peaks (budesonide epimers A and B) in the chromatogram.

The following pages illustrate that the acceptance criteras are being met despite Impurity A and epimers of impurity K are not identified in the given chromatogram (not present in tested sample). Thus using a Purospher® STAR RP-18 endcapped (3 μm), Hibar® RT 150x4.6 mm column it is possible to comply with the given requirement for the analysis of Budesonide and related substancesthe given requirement for the analysis of Budesonide and related substances.


Chromatographic ConditionsColumn: Purospher® STAR RP-18 endcapped (3 μm) Hibar®150x4.6 mm 1.50470.0001

Budesonide and Related Substances (EP)Purospher® STAR RP-18 endcapped

Column: Purospher STAR RP 18 endcapped (3 μm) Hibar 150x4.6 mm 1.50470.0001Injection: 20 μLDetection: UV, 240 nmCell: 10 μLFlow Rate: 1.0 mL/min

Mobile Phase:

Buffer: to 900 mL of a 4 g/L solution of sodium dihydrogen phosphate , add 100 mL of a 2.5 g/L solution of phosphoric acid . Adjust the pH if necessary.A: Anhydrous ethanol , acetonitrile, phosphate buffer solution pH 3.2; 2:32:68 (v/v/v)B: Acetonitrile, phosphate buffer solution pH 3.2 R; 50:50 (v/v), p p p ; ( / )

Gradient: See tableTemperature: 50˚CDiluent: Acetonitrile R, phosphate buffer solution pH 3.2; 32:68 (v/v)Test Solution: 50 mg of the substance to be examined in 15 mL of acetonitrile and dilute to final volume

(50 mL) with phosphate buffer solution pH 3.2.Resolution Solution: Dilute 1.0 mL of test solution (a) to 10.0 mL with the solvent mixture.

Dilute 1.0 mL of this solution to 100.0 mL with the solvent mixture.Pressure Drop: 182 - 162 Bar (2639 - 2349 psi)

Time (min)

A(%)

B(%)

0.0 100 0

38.0 100 0

50.0 0 1002

3

4

5

ensi

ty (m

V) Blank

60.0 0 100

65.0 100 0

70.0 100 0

4

5

V)

0

1

0 5 10 15 20 25 30 35 40

Int

Retention (min)

E i B E i A

0

1

2

3

0 5 10 15 20 25 30 35 40

Inte

nsit

y (m

V

SST solutionEpimer B Epimer A



Budesonide and Related Substances (EP)Purospher® STAR RP-18 endcapped

8

10

y D

mpu

rity

L

G

22R- and 22S-Budesonide

2

4

6

Inte

nsit

y (m

V)

Test solution

Impu

rity

DEp

imer

of Im

purit

y

Im

Impu

rity

GEp

imer

of Im

purit

y

0

2

0 5 10 15 20 25 30 35 40


I thi l ith i it A th i f i it K ld b f d!

Chromatographic Data:

No. Compound Retention Time (min) Resolution RRT

1 Impurity D 11.4 0.66

Suitability requirement: NLT 1.5

In this sample neither impurity A, nor the epimers of impurity K could be found!

1 Impurity D 11.4 0.66

2 Epimer of Impurity D 12.0 0.69

3 Impurity L 15.4 0.90

4 Budesonide Epimer B 17.2 1.00

5 Budesonide Epimer A 18.4 2.2 1.07

6 Impurity G 21.0 1.22

7 Epimer of Impurity G 22 6 1 317 Epimer of Impurity G 22.6 1.31


Dipyridamole and Related Substances (EP)

Dipyridamole

Dipyridamole inhibits thrombus formation when given chronically and causes vasodilatation when given at high doses over a short time. Common tradename is Persantine.

The current EP monograph method for Dipyridamole and related substances specifies the use of a 100x4.0 mm column with end-capped octadecylsilyl (ODS or RP-18) silica gel for chromatography R (5 μm). The relative retention with reference to dipyridamole (retention time = about 8 min); impurity B = about 0.2; impurity F = about 0.3; impurity D = about 0.9; impurity E = about 1.3; impurity C = about 1.6;impurity A = about 2.2.– The system suitability criteria also specify a minimum resolution of 2.0 between the peaks due to impurity D and dipyridamole.

The c rrent EP method specifies the se of a 100 4 0 mm col mn ith 5 m RP 18 stationar phase b tThe current EP method specifies the use of a 100x4.0 mm column with 5 μm RP-18 stationary phase, but we used a 100x4.6 mm column instead (allowed change). Due to the large column tube volume we added another six minutes for column re-equilibration which explains why total method run-time is six minutes longer.

The following pages illustrate that the acceptance critera are being met and it is possible to use a 100x4.6 mm Purospher® STAR RP-18 endcapped (5 μm) column to comply with the given requirement.


Column: Purospher® STAR RP-18 endcapped (5μm) Hibar® RT 100x4.6 mm 1.50622Injection: 5 μL

Dipyridamole and Related Substances (EP)Purospher® STAR RP-18 endcapped

j μDetection: UV 295 nmCell: 10 μLFlow Rate: 1.2 mL/minMobile Phase: A: dissolve 1.0 g of potassium dihydrogen phosphate R in 900 mL of to water

adjust to pH 7.0 with 0.5 M sodium hydroxide and dilute 1000 mL with water.B: Methanol

Gradient: See tableTemperature: 45˚C, Sample cooler at 10˚CDiluent: MethanolSample Solution: Dissolve 0.100 g of the substance to be examined in methanol and dilute to 50 mL with the same. Reference Solution: Dilute 1.0 mL of the test solution to 100.0 mL with methanol.

Dilute 1.0 mL of this solution to 10.0 mL with methanolPressure Drop: 101 - 56 Bar (1464 -812 psi)

Time A B2

4

6

8

Inte

nsit

y (m

V)

Standard Solution

(min) (%) (%)

0.0 40 60

5.0 40 60

19.0 5 95

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Blank


38


Dipyridamole and Related Substances (EP)Purospher® STAR RP-18 endcapped

4

6

8

10

ensi

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V)

Sample solutionrity

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rity

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mpu

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Impu

rity

A

0

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4

0 5 10 15 20 25 30 35

Int


p

Impu Im


No. Compound Retention Time (min) Resolution Observed RRT RRT per EP

1 Impurity B 2.2 0.23 About 0.2

2 Impurity F 2.5 0.26 About 0.3

3 Impurity D 8.8 0.93 About 0.9

4 Dipyridamole 9 5 3 3 1.00 About 1.0

mAUPeakPurity Curve

Peak Purity curve UV spectramAU

4 Dipyridamole 9.5 3.3 1.00 About 1.0

5 Impurity E 12.5 1.31 About 1.3

6 Impurity C 16.7 1.75 About 1.6

7 Impurity A 18.6 1.96 About 2.2

0.00

0.25

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256

348

289

9.3 9.4 9.5 9.6 min0.0

200 250 300 350nm

39


Dofetilide and Related Substances (USP)

Dofetilide

Dofetilide is a class III antiarrhythmic agent. It is marketed under the trade name Tikosyn. Due to the pro-arrhythmic potential, it is only available by prescription by physicians who have undergone specific training in the risks of treatment with dofetilide.

The current USP monograph method for Dofetilide and related substances specifies the use of a 150x3 9The current USP monograph method for Dofetilide and related substances specifies the use of a 150x3.9 mm column with and L1 (ODS or RP-18) packing having 5 μm particle size. System suitability requirements; a resolution of NLT 5.0 between dofetilide and dofetilide related compound A; a column efficiency of NLT 35,000 theoretical plates for the dofetilide peak; and with a tailing factor of NMT 1.5 for the dofetilide peak (using the system suitability solution).

We have redeveloped this monograph method using a monolithic Chromolith® HighResolution RP-18 p g p g gendcapped column and the following pages illustrate that the all acceptance critera are being met and the new method provide excellent performance at very low system pressure.

Using a monolithic column over a particulate counterpart, additional column life-time can be expected due to the more robust stationary phase backbone. In addtion, a monolithic column exhibit lower backpressure and thus less wear on HPLC system (extra safety and lower maintenance cost). In summary, this means an overall lower cost per analysis.


Dofetilide and Related Substances (USP)Chromolith® HighResolution RP-18 endcappedChromatographic ConditionsColumn: Chromolith® HighResolution RP 18 endcapped 100x4 6 mm 1 52022 0001Column: Chromolith® HighResolution RP-18 endcapped, 100x4.6 mm 1.52022.0001Injection: 20 μLDetection: UV 230 nmCell: 10 μLFlow Rate: 1.0 mL/minMobile Phase: A: 0.78 g/L of ammonium acetate. Adjust with glacial acetic acid to a pH of 5.0 ± 0.1

B: MethanolGradient: See tableTemperature: 30˚CDiluent: Acetonitrile and Buffer; 3:22 (v/v)SST Solution: 1.25 μg/mL each of Dofetilide and Dofetilide Related Compound A in diluentStandard Solution: 1.25 μg/mL of USP Dofetilide in DiluentSample soltion 0.25 mg/mL of Dofetilide in DiluentPressure Drop: 52-65 Bar (754 - 943 psi)

Time (min)

A(%)

B(%)20

40

60

80

100

Inte

nsit

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V)

SST solution

Dof

etili

de R

C A

( ) ( ) ( )

0.0 88 12

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Dofetilide and Related Substances (USP)Chromolith® HighResolution RP-18 endcapped

100

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40

60

80

Inte

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V) Sample solution

With the use of a Chromolith® HighResolution column you can meet the system suitability

60

80

100

(mV)

Standard solution

00 5 10 15 20 25 30 35 40


without changing any parameter besides a reduction in injection volume from 50 to 20 μL.

New method would require revalidation though because of specification in monograph of

0

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0 5 10 15 20 25 30 35 40

Inte

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specification in monograph of column with particle size.

( )


No. Compound Retention Time (min) Resolution Theoretical Plate

1 Dofetilide Related compound A 14.2 699682 Dofetilide 15.8 7.7 83075

Parameter USP requirement Observed value

Resolution 5.0 7.7

Theoretical plate for Dofetilide peak 35000 83075

Tailing factor for Dofetilide peak NMT 1.5 1.1


Domperidone and Related Substances (EP)

Domperidone

Domperidone is a peripheral, specific blocker of dopamine receptors. Domperidone is given in order to relieve nausea and vomiting; to increase the transit of food through the stomach (as a prokinetic agent through increase in gastrointestinal peristalsis); and to increase lactation (breast milk production) by release of prolactin. Common trade names are: Motilium, Motillium, Motinorm Costi, Nomit and Molax.

The current EP monograph method for Domperidone and related substances specifies use of a 100x4.6 mm column with base-deactivated octadecylsilyl (ODS or RP-18) silica gel for chromatography R (3 μm) as stationary phase. System suitability requirements specify the relative retention with reference to domperidone (retention time = about 6.5 min): impurity A = about 0.4; impurity B = about 0.65; impurity C b t 0 7 d id l b t 1 1 i it D b t 1 15 i it E b t 1 2 i it FC = about 0.7; droperidol = about 1.1; impurity D = about 1.15; impurity E = about 1.2; impurity F = about 1.3.

The following pages illustrate that the acceptance critera are being met for Domperidone by following the current EP monograph. Thus using a Purospher® STAR RP-18 endcapped (3 μm), Hibar® RT 100x4.6 mm column it is possible comply with the requirement for Domperidone and related substances analysis.


Chromatographic Conditions:Column: Purospher® STAR RP-18 endcapped (3μm) Hibar® RT 100x4.6 HPLC column 1.50469.0001

Domperidine and Related Substances (EP)Purospher® STAR RP-18 endcapped

Column: Purospher STAR RP 18 endcapped (3μm) Hibar RT 100x4.6 HPLC column 1.50469.0001Injection: 10 μLDetection: UV, 284 nmCell: 10 μLFlow Rate: 1.5 mL/minMobile Phase: Solution A: 5 g/L solution of ammonium acetate

Solution B: MethanolGradient: See tableTemperature: 25 °CpDiluent: DimethylformamideRefrence Solution: Dilute 1.0 mL of the test solution to 100.0 mL with dimethylformamide.

Dilute 5.0 mL of this solution to 20.0 mL with dimethylformamide.Test Solution: Dissolve 0.10 g of the domperidone in dimethylformamide and dilute to 10.0 mL

with the same solvent.Pressure Drop: 260 - 104 Bar (3770 - 1508 psi)

Time (min)

A(%)

B(%)100

200

300

400

500

Inte

nsit

y (m

V)

Standard

( ) ( ) ( )

0.01 70 30

10.0 0 100

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Retention time (min)Retention time (min)


Domperidine and Related Substances (EP)Purospher® STAR RP-18 endcapped

120

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Sample

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Impu

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purit

y F

0

30

0 5 10 15


No impurity B (RRT of about 0.65) nor impurity D (RRT of about1.15) could be identified in this sample

Chromatographic Data : Sample

No. Compound Retention Time (min) Resolution Observed RRT RRT Specification (EP)

No impurity B (RRT of about 0.65) nor impurity D (RRT of about1.15) could be identified in this sample

1 Impurity A 3.4 0.44 About 0.42 Impurity C 5.7 0.75 About 0.73 Domperidone 7.6 1.004 Droperidole 8.5 6.0 1.11 About 1.15 Impurity E 9.4 1.23 About 1.26 Impurity F 9.6 1.27 About 1.3p y


Fluvoxamine and Related Substances (USP)

Fluvoxamine

Fluvoxamine is a medication which functions as a selective serotonin reuptake inhibitor (SSRI) and σ1 receptor agonist. Fluvoxamine is used primarily for the treatment of obsessive-compulsive disorder (OCD), and is also used to treat major depressive disorder (MDD), and anxiety disorders such as panic disorder and posttraumatic stress disorder (PTSD).Fluvoxamine CR (controlled release) is approved to treat social anxiety disorder. Common commercial brand names: Floxyfral, Luvox, Fevarin.

The current USP monograph method for Fluvoxamine maleate and related substances specifies the use of a 250x4.6 mm column with L7 (RP-8) packing as stationary phase (no particle size mentioned), and with identical experimental conditions as described in the assay method. System suitability requirements for related substances are provided by means of relative retention time (RRT) for identified and known imp rites see tab lated imp rites in chromatograph performance table In addition for the assaimpurites, see tabulated impurites in chromatography performance table. In addition, for the assay method, the column efficiency should be not less than 5000 theoretical plates and the tailing factor is not more than 2.0.

The following pages illustrate that the acceptance critera are being met for the Fluvoxamine maleate and related substances methods by following the current USP monograph and using identical matched column. Using a 250x4.6 mm Purospher® STAR RP-8 endcapped (5 μm) column it is possible to comply with the requirement for Fluvoxamine maleate and related substances analysis.with the requirement for Fluvoxamine maleate and related substances analysis.


Chromatographic ConditionsColumn: Purospher® STAR RP 8 endcapped (5μm) Hibar® 250x4 6 mm 1 51454 0001

Fluvoxamine and Related Substances (USP)Purospher® STAR RP-8 endcapped

Column: Purospher® STAR RP-8 endcapped (5μm) Hibar® 250x4.6 mm 1.51454.0001Injection: 20 μLDetection: UV 234 nmCell: 10 μLFlow Rate: 1.7 mL/minMobile Phase: Buffer: dissolve about 5 g of 1-pentanesulfonic acid sodium salt and 0.7 g of monobasic

potassium phosphate in 620 mL of water. Adjust with phosphoric acid to a pH of 3.00 ± 0.05. Mix buffer and acetonitrile 62:38 (v/v)bu e a d aceto t e 6 38 ( / )

Temperature: 40 °CDiluent: Mobile phaseResolution Solution: Transfer about 6 mg of Fluvoxamine Maleate to a 50-mL volumetric flask. Heat the sample at

120 for 10 minutes. Cool down to room temperature, add 3.0 mL of 0.1 N hydrochloric acid. Heat the solution in a water bath for 10 minutes. Cool down to room temperature, add 50 mg of Fluvoxamine Maleate, and dissolve in 25 mL of Mobile phase. Dilute with Mobile phase to volume, and mix.

P D 179 B (2596 i)Pressure Drop: 179 Bar (2596 psi)

250

300

100

150

200

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Blank

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-Met

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me

Ma

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etho

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none

oxResolution solution


00 10 20 30 40 50


5-


No. Compound Retention Time (min)

Resolution Observed RRT Approx RRT USP guideline

1 Maleic acid 1.6 0.17 0.19

2 5-Methoxy Oxime 4.9 0.54 0.50

3 Z- Isomer 7.5 11.6 0.82 0.79

44 Fluvoxamine 9.2 5.1 1.00 1.0

5 4-Trifluoromethyl oxime Maleate 18.6 2.02 2.00

6 5-methoxy valerophenone oxime 29.7 3.23 3.45

In the analyzed samples neither: 5-Methoxy-4¢-(trifluoromethyl)valerophenone(E)-O-(2-aminoethyl)aminoethyl oxime maleate (RRT 0.67); 4¢-(Trifluoromethyl)valerophenone(E)-O-2-(2-aminoethyl)aminoethyl oxime maleate (RRT 1.18); (E)-O-2-(2-Aminoethyl)-4-(trifluoromethyl)- -phenylacetophenone oxime maleate (RRT 1.74) nor y y p y p5-Methoxy-4¢-(trifluoromethyl)valerophenone ketone (RRT 4.2) could be found!



UV spectra: Fluvoxamine Peak purity curve: Fluvoxamine

2000

2500

mAU

0 50

0.75

500

750

mAU

PeakPurity Curve

200 250 300 350 nm

0

500

1000

1500

22

4

35

2

25

1

9.00 9.25 9.50 9.75 min

0.00

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Zero Line

UV spectra: Z-isomer Peak purity curve: Z-isomer

1000

1250

mAU

0 75

400

mAU

PeakPurity Curve

0

250

500

750

1000

332

0.00

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Zero Line

200 250 300 350 nm 7.25 7.50 7.75 8.00 min0


Pramipexole and Related Substances (USP)Purospher® STAR RP-18 endcapped

Pramipexole

Pramipexole is a dopamine agonist used for treating Parkinson's disease (PD) and restless legs syndrome (RLS) Common brand names: Mirapex Mirapexin Sifrolsyndrome (RLS). Common brand names: Mirapex, Mirapexin, Sifrol.

The current USP monograph method for Olanzapine and related substances as well as for the assay method specifies th use of a 150x4.6 mm column with 5 μm L1 (RP-18) packing as stationary phase. System suitability requirements for related substances are; Resolution: NLT 6.0 between pramipexole related compound A and pramipexole; and a tailing factor NMT 2.0 for pramipexole. In addition, in the assay method the relative retention times for pramipexole related compound A and pramipexole are specified about 0.7 and 1.0, respectively.

The following pages illustrate that the acceptance critera are met for both the Pramipexole and related substances as well as the assay method by following the current USP monograph using a 150x4.6 mm Purospher® STAR RP-18 endcapped (5 μm) column.

50


Chromatographic ConditionsColumn: Purospher® STAR RP-18 endcapped (5 μm) Hibar® 150x4.6 mm 1.51455.0001


Column: Purospher STAR RP 18 endcapped (5 μm) Hibar 150x4.6 mm 1.51455.0001Injection: 5 μLDetection: Shimadzu Prominence, UV 264 nmCell: 10 μLFlow Rate: 1.5 mL/minMobile Phase: Solution A: Dissolve 9.1 g of potassium dihydrogen phosphate and 5.0 g of sodium

1- octanesulfonate monohydrate in 1 L of water. Adjust with phosphoric acid to a pH of 3.00 ± 0.05. Solution B: Acetonitrile and Solution A; 1:1 (v/v); ( / )

Gradient: See tableTemperature: 45 °CDiluent: Acetonitrile and Solution A; 1:4 (v/v)Resolution Solution: 1.5 μg/mL of USP Pramipexole Dihydrochloride in DiluentSample solution: 1.5 mg/mL of Pramipexole Dihydrochloride in DiluentPressure Drop: 209 - 215 Bar (3030 - 3117 psi)

Time ( i )

A(%)

B(%)

5

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15

Inte

nsit

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V) Standard

(min) (%) (%)

0.0 60 40

15.0 20 80

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51



25

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rop


No. Compound Retention Time (min) Resolution Tailing Factor RRT

1 Impurity A 4.3 0.71

2 Pramipexole 6.0 13.9 1.00 1.00

3 N-Propylpramipexole 8.8 1.473 py p p

UV spectra - Pramipexole Peak purity curve –Pramipexole

750

mAU

26

4

0 75

1.00

1000

1250

mAU

Peak

Purity Curve

200.0 225.0 250.0 275.0 300.0 325.0 350.0 nm

0

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22

9

20

6

34

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19

4

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5.75 6.00 6.25 6.50 min

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Zero Line

52

200.0 225.0 250.0 275.0 300.0 325.0 350.0 nm


A transfer of HPLC methods to UHPLC requires scaling down from bigger to smaller inner diameter (e.g. 4.6 2.1 mm i.d.) and from long to short columns (e.g. 250/150 to 100 or 50 mm length) in addition to the reduction of particle sizes (from e.g. 5 μm to 2 μm). To ensure equivalent chromatographic separation it is also necessary to scale the flow rate injection volume and the gradient parameters

From HPLC to UHPLC

separation, it is also necessary to scale the flow rate, injection volume and the gradient parameters.

Column length L L x dp / dpL1 - HPLC column lengthL2 - UHPLC column length

Adjusting the column lengthThe first step is to determine the appropriate column length in order to maintain the same separation. Keeping the same column length while decreasing the particle size will increase the number of theoretical plates as well as the backpressure. Therefore, when decreasing particle size, column length can be shortened without losing resolution.

Scaling the flow rateDecreasing the internal diameter of the column (e.g. from 4.6 to 2.1 mm) requires recalculating column flow rate in order to maintain same linear velocity. Linear velocity is defined as the distance which mobile phase travels over time (cm/min), whereas flow rate is the volume of mobile phase that travels over time (mL/min). To maintain the same linear velocity through a column with a smaller internal diameter, the flow rate must be decreased proportionally to the column internal diameter according to the equation

Column length L2 = L1 x dp2 / dp1 dp1 - HPLC particle sizedp2 - UHPLC particle size

flow rate must be decreased proportionally to the column internal diameter according to the equation below.

Flow rate f2 = f1 x (d2)2 / (d1)2f1 - HPLC flow ratef2 - UHPLC flow rate (mL/min)d1 - HPLC column ID d2 - UHPLC column ID (mm)

Scaling the injection volumeDecreasing the column internal diameter and length, decreases the overall column volume and sample capacity. Therefore, we must alter the injection volume. Please note that since overall column volume has p y jdecreased, it is more important to match the sample solvent to the starting mobile phase composition. Mismatched sample solvents can cause irreproducible retention times, efficiencies, and even changes in selectivity. If using a larger injection volume than calculated, check for peak abnormalities and irreproducibility that could result from phase overload.

Injection volume V2 = V1 x (d22/d1

2) x (L2 / L1)

V1 - HPLC Injection volumeV2 - UHPLC Injection volumed1 - HPLC column ID d2 - UHPLC column ID (mm)L HPLC l l thL1 - HPLC column length L2 - UHPLC column length

Adjusting gradient timeWhen an analytical method is scaled down, the time program of the gradient also needs to be scaled down to keep the gradient volume the same.

Time: t2 = t1 x (f1/f2) x (d2²/d1²) x (L2 / L1)

t1 - HPLC timet2 - UHPLC timef1 - HPLC flow ratef2 - UHPLC flow rate (mL/min)L1 - HPLC column length

53

L2 - UHPLC column length


Fexofenadine and Related Substances (USP)

Fexofenadine

Fexofenadine is an antihistamine pharmaceutical drug used in the treatment of allergy symptoms, such as hay fever, nasal congestion, and urticaria. Common trade names are: Allegra, Fexidine, Telfast, Fastofen, Tilfur, Vifas, Telfexo, Allerfexo.

The current USP monograph method for Fexofenadine and related substances specifies the use of a 250x4.6 mm column with L11 (Phenyl) packing as stationary phase, which is identical to conditions in the assay method also No particle size mentioned wherefore the ratio must be calculated using the largestassay method also. No particle size mentioned wherefore the ratio must be calculated using the largest particle size consigned in the USP definition of the column.

System suitability requirements specify a chromatographic resolution not less than (NLT) 10 between fexofenadine and fexofenadine related compound A; a peak tailing factor of not more than (NMT) 2.0.

The following pages illustrate that the acceptance criteras are being met for Fexofenadine assay and its related substances methods by following the current USP monograph and using identical matched column. Using a 250x4.6 mm Purospher® STAR Phenyl (5 μm) column you can comply with the requirements for Fexofenadine assay and its related substances analysis.

To address the current trend of monograph modernization, and improving the method in terms of selectivity, speed and sensitivity, we also scaled this method to a 100x2.1 mm UHPLC column; Purospher® STAR Phenyl (2 μm). The acceptance critera are met for the for Fexofenadine monograph. This is however, a non-allowed scaling and would require a complete revalidation of the new method for approvalapproval.


Fexofenadine and Related Substances (USP)Purospher® STAR Phenyl - HPLCChromatographic ConditionsColumn: Purospher® STAR Phenyl (5μm) Hibar® RT 250x4 6 HPLC column 1 51918 0001Column: Purospher® STAR Phenyl (5μm) Hibar® RT 250x4.6 HPLC column 1.51918.0001Injection: 20 μLDetection: UV 220 nmCell: 10 μLFlow Rate: 1.5 mL/minMobile Phase: Buffer: 6.64 g/L of monobasic sodium phosphate and 0.84 g/L of sodium perchlorate in water.

Adjust with phosphoric acid to a pH of 2.0. Mix acetonitrile and buffer 7:13 (v/v). Add 3 mL/L of triethylamine.y

Temperature: 25˚CDiluent: Acetonitrile and Buffer 1:1 (v/v)SST Solution: 0.06 mg/mL of Fexofenadine Hydrochloride RS and

0.005 mg/mL of Fexofenadine Related Compound A in Mobile phaseSample Solution: 1.0 mg/mL of Fexofenadine Hydrochloride in DiluentPressure Drop: 164 Bar (2378psi)

S l l ti50 Sample solution

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Blank

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Fexofenadine and Related Substances (USP)Purospher® STAR Phenyl - HPLC

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Suitability requirement: NLT 10 in resolutionNMT 2.0 in Tailing factor

No. Compound Retention Time (min) Resolution Tailing Factor

1 Fexofenadine 10.3 1.1

2 Related compound A 16.3 10.9 1.0

Recommended chemicals and reagents

Name of the chemical Article Number

1 Sodium dihydrogen phosphate dihydrate for analysis EMSURE® Reag. Ph Eur 1.06342

2 Sodium perchlorate monohydrate for analysis EMSURE® 1.06564

3 ortho-Phosphoric acid 85% for analysis EMSURE® ACS,ISO,Reag. Ph Eur 1.00573

4 Acetonitrile gradient grade for liquid chromatography LiChrosolv® Reag Ph Eur 1 000304 Acetonitrile gradient grade for liquid chromatography LiChrosolv Reag. Ph Eur 1.00030


Chromatographic ConditionsC l P h ® STAR Ph l (2 ) Hib ® HR 100 2 1 1 51014 0001

Fexofenadine and Related Substances (USP)Purospher® STAR Phenyl - UHPLC

Column: Purospher® STAR Phenyl.(2 μm) Hibar® HR 100x2.1 mm 1.51014.0001Injection: 2 μLDetection: UV 220 nmCell: 2.5 μLFlow Rate: 0.4 mL/minMobile Phase: 6.64 g/L of monobasic sodium phosphate and 0.84 g/L of sodium perchlorate in water.

Adjust with phosphoric acid to a pH of 2.0. Mix Acetonitrile and buffer 7:13 (v/v) Add 3 mL/L of triethylamineMix Acetonitrile and buffer 7:13 (v/v). Add 3 mL/L of triethylamine.

Temperature: 25˚CDiluent: Acetonitrile and Buffer 1:1 (v/v)SST Solution: 0.06 mg/mL of Fexofenadine Hydrochloride RS and

0.005 mg/mL of Fexofenadine Related Compound A in mobile phaseSample Solution: 1.0 mg/mL of Fexofenadine Hydrochloride in DiluentPressure Drop: 388 Bar (5626psi) Scaling 250x4.6 to 100x2.1

20

40

60

80

100

Inte

nsit

y (m

V)

Sample solution Column volume reduction: 12X (and thereby injection volume also)

Flow rate reduction: 12X (to maintain same retention time)

Flow rate reduction: 4.8 X(to maintain same linear velocity)

6

8

10

(mV)

Blank

00 3 6 9 12 15


(to maintain same linear velocity)

Injection volume should be:20/12= 1.67 but we used 2 μL (more practical)

Flow rate should either be 0.115 or 0.315 mL/min. We used 0.4 mL/min to optimize analysis time and still comply with system suitability criterias

0

2

4

6

0 3 6 9 12 15

Inte

nsit

y


with system suitability criterias

Flowrate per USP 37 change guidelines

F2=1.5 x (2.12x5)/(4.62’2) = 0.78 mL/min

This would be completely impractical and backpressure would be beyond tolerance of column and system.


y

Fexofenadine and Related Substances (USP)Purospher® STAR Phenyl - UHPLC

45

60

mV)

15

30

Inte

nsit

y (m

Resolution solution

Rela

ted

com

poun

d A

00 3 6 9 12 15



Suitability requirement: NLT 10 in resolutionNMT 2.0 in Tailing factor


No. Compound Retention Time (min) Resolution Tailing Factor

1 Fexofenadine 3.7 1.0

2 Related compound A 6.0 12.7 1.0

Recommended chemicals and reagents

Name of the chemical Article Number

1 Sodium dihydrogen phosphate dihydrate for analysis EMSURE® Reag. Ph Eur 1.06342

2 Sodium perchlorate monohydrate for analysis EMSURE® 1.06564

3 ortho-Phosphoric acid 85% for analysis EMSURE® ACS,ISO,Reag. Ph Eur 1.00573

4 Acetonitrile gradient grade for liquid chromatography LiChrosolv® Reag Ph Eur 1 00030


4 Acetonitrile gradient grade for liquid chromatography LiChrosolv Reag. Ph Eur 1.00030

Olanzapine and Related Substances (USP)Purospher® STAR RP-8 endcapped

Olanzapine

Olanzapine is an atypical antipsychotic, approved for the treatment of schizophrenia and bipolar disorder. Common brand names: Zyprexa, Zalasta, Zolafren, Olzapin, Oferta, Zypadhera.Combination Drug: Symbyax (Olanzapine and Fluoxetine)

The current USP monograph method for Olanzapine and related substances specifies use of a 250x4.6 mm column with 5 μm L7 (RP-8) packing as stationary phase. S t it bilit i t f l t d b t th l ti b t Ol i dSystem suitability requirements for related substances are; the resolution between Olanzapine and Olanzapine related substance A should not be less than 3.0 and the tailing factor should not be more than 1.5 for the Olanzapine peak.

The following pages illustrate that the acceptance critera are being met for the Olanzapine and related substances method by following the current USP monograph using a 250x4.6 mm Purospher® STAR RP-8 endcapped (5 μm) column.

To address the current trend of monograph modernization, and improving the method in terms of selectivity, speed and sensitivity, we also scaled this method to a 100x2.1 mm UHPLC column; Purospher® STAR RP-8 endcapped (2 μm). Also in this example, all the system suitability critera are met for the Olanzapine and related substances. This is however, a non-allowed scaling and would require a complete revalidation of the new method for approval. For gradient separations, changes in length, column inner diameter and particle size are not allowed. This is new in USP 37.g , p

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Chromatographic ConditionsColumn: Purospher® STAR RP-8 endcapped (5μm) Hibar® 250x4.6 mm 1.51454.0001

Olanzapine and Related Substances (USP)Purospher® STAR RP-8 endcapped - HPLC

Column: Purospher STAR RP 8 endcapped (5μm) Hibar 250x4.6 mm 1.51454.0001Injection: 20 μLDetection: UV 220 nmCell: 10 μLFlow Rate: 1.5 mL/minMobile Phase: Buffer: Dissolve 13 g of sodium dodecyl sulfate in 1500 mL of water. Add 5 mL of phosphoric acid,

and adjust with a sodium hydroxide solution to a pH of 2.5.A: Acetonitrile and Buffer 48:52 (v/v)B: Acetonitrile and Buffer 70:30 (v/v)( / )

Gradient: See tableTemperature: 35˚C, Autosampler cooler at 5˚CDiluent: 37 mg/L of EDTA disodium in Buffer. Acetonitrile and EDTA solution 40:60 (v/v)Test Solution: 20 μg/mL of USP Olanzapine, and 2 μg/mL each of Related Compound A and B in diluentStandard Solution: 2 μg/mL of USP Olanzapine in diluentSample Solution: 0.4 mg/mL of Olanzapine in diluentPressure Drop: 168 - 129 Bar (2434 - 1871 psi)

Time(min)

A(%)

B(%)

0 0 100 0

20

30

40

50

tens

ity

(mV)

Standard solution

0.0 100 0

8.0 100 0

17.0 70 30

20.0 70 30

20.1 100 0

23.0 100 0

0

10

0 5 10 15 20 25 30 35

In


40

50

mV)

0

10

20

30

0 5 10 15 20 25 30 35

Inte

nsit

y (m


Blank

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Olanzapine and Related Substances (USP)Purospher® STAR RP-8 endcapped - HPLC

80

100

SST solution

40

60

Inte

nsit

y (m

V)

Impu

rity

B

Impu

rity

A

Tailing Factor: 1.01

0

20

0 5 10 15 20 25 30 35




1 Olanzapine Related compound B 3.9 0.21

2 Olanzapine Related compound A 10.9 34.3 0.59

3 Olanzapine 18 6 37 9 1 003 Olanzapine 18.6 37.9 1.00

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Chromatographic ConditionsColumn: Purospher® STAR RP-8 endcapped (2μm) Hibar® HR 100x2.1 mm 1.50629.0001

Olanzapine and Related Substances (USP)Purospher® STAR RP-8 endcapped - UHPLC

Column: Purospher STAR RP 8 endcapped (2μm) Hibar HR 100x2.1 mm 1.50629.0001Injection: 5 μL (larger injection volume than according to column tube volume reduction)Detection: UV 220 nmCell: 2.5 μLFlow Rate: 0.5 mL/min (faster flow rate – thus not according to same linear velocity)Mobile Phase: Buffer: Dissolve 13 g of sodium dodecyl sulfate in 1500 mL of water. Add 5 mL of phosphoric

acid, and adjust with a sodium hydroxide solution to a pH of 2.5.A: Acetonitrile and Buffer 48:52 (v/v)B: Acetonitrile and Buffer 70:30 (v/v)( / )

Gradient: See tableTemperature: 35˚C, Sample cooler at 5˚CDiluent: 37 mg/L of EDTA disodium in Buffer. Acetonitrile and EDTA solution 40:60 (v/v)Test Solution: 20 μg/mL of USP Olanzapine, and 2 μg/mL each of Related Compound A

and Related Compound B in diluentStandard Solution: 2 μg/mL of USP Olanzapine in diluentSample Solution: 0.4 mg/mL of Olanzapine in DiluentPressure Drop: 408 - 311 Bar (5916 - 4510 psi)

Time(min)

A(%)

B(%)

0.0 100 0

2.35 100 0

4.85 0 100

6 1 0 100

8

12

16

20

nten

sity

(mV)

Standard

6.1 0 100

6.6 100 0

9.0 100 0

16

20

mV)

0

4

0 2 4 6 8

In


Scaling 250x4.6 to 100x2.1

0

4

8

12

0 2 4 6 8

Inte

nsit

y (m


BlankColumn volume reduction: 12X (and thereby injection volume also)

Flow rate reduction: 12X (to maintain same retention time) or

Flow rate reduction: 4.8 X(to maintain same linear velocity)

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Olanzapine and Related Substances (USP)Purospher® STAR RP-8 endcapped - UHPLC

100

40

60

80

nten

sity

(mV)

Sample solution

0

20

0 2 4 6 8

In

Retention time (min)150

60

90

120

150

tens

ity

(mV)

SST Solution

ty A

ty B

T ili F t 1 10

0

30

60

0 2 4 6 8

Int


Impu

ri

Impu

ri Tailing Factor: 1.10



1 Olanzapine Related compound A 1.0 0.19

2 Olanzapine Related compound B 3.0 26.1 0.55

3 Olanzapine 5.4 33.6 1.00

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Rizatriptan and Related Substances (USP)Purospher® STAR Phenyl

Rizatriptan is a 5-HT1 receptor agonist for the treatment of migraine headaches.Common brand name is Maxalt.

Rizatriptan

The current USP monograph method for Rizatriptan and related substances as well as the assay method specifies the use of a 250x4.6 mm column with 5 μm L11 (Phenyl) packing as stationary phase. System suitability criteria in the assay method specify that the relative retention times for rizatriptan and benzoic acid are 1.0 and about 2.1, respectively., as well as a tailing factor NMT 3.5 for rizatriptan. System suitability requirements for related substances are; the relative retention times for rizatriptan, rizatriptan impurity C, and benzoic acid are 1.0, about 1.3, and p , p p y , , ,about 2.1 RRT. The resolution is NLT 2.0 between rizatriptan and rizatriptan impurity C; and using the sensitivity solution the signal-to-noise ratio should be NLT 10 for the rizatriptain peak.

The following pages illustrate that the acceptance criteras are met for the assay and there is higher resolution and retention for the impurites in the Rizatriptan and related substances method by following the current USP monograph using a 250x4.6 mm Purospher® STAR Phenyl (5 μm) column(5 μm) column.

To address the current trend of monograph modernization, and improving the method in terms of selectivity, speed and sensitivity, we also scaled this method to a 100x2.1 mm UHPLC column; Purospher® STAR Phenyl (2 μm). Also in this example, all the system suitability critera are met. This is however, a non-allowed scaling and would require a complete revalidation of the new method for approval. For gradient separations, changes in length, column inner diameter and

ti l i t ll d Thi i i USP 37particle size are not allowed. This is new in USP 37.

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Chromatographic ConditionsColumn: Purospher® STAR Phenyl (5μm) Hibar® RT 250x4 6 mm 1 51918 0001

Rizatriptan and Related Substances (USP)Purospher® STAR Phenyl - HPLC

Column: Purospher® STAR Phenyl (5μm) Hibar® RT 250x4.6 mm 1.51918.0001Injection: 20 μLDetection: UV 280 nmCell: 10 μLFlow Rate: 1.5 mL/minMobile Phase: A: 1.0 mL of trifluroacetic acid to 1 L of a solution of acetonitrile and Water 4:21 (v/v), and mix.

B: Acetonitrile and trifluroacetic acid 1000:1 (v/v) (or use our premixed product 4.80448)Gradient: See tableTemperature: 40˚CTemperature: 40 CDiluent: Solution ATest Solution: 1 mg/mL of USP Rizatriptan Benzoate System Suitability Mixture in Solution AResolution Solution: 0.5 μg/mL of Rizatriptan Benzoate obtained by suitable dilution of the Sample

solution with Solution APressure Drop: 140 - 129 Bar (2030 - 1871 psi)

250

Time(min)

A(%)

B(%)

0.0 100 050

100

150

200

Inte

nsit

y (m

V)

SST solutionImpu

rity

C

8.0 100 0

17.0 70 30

20.0 70 30

20.1 100 0

23.0 100 0

00 5 10 15 20 25


150

200

250

y (m

V)

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0 5 10 15 20 25

Inte

nsit

y


Blank

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Rizatriptan and Related Substances (USP)Purospher® STAR Phenyl - HPLC

5

6

7

V)

Sensitivity solution

1

2

3

4

Inte

nsit

y (m

V


0

1

0 5 10 15 20 25


USP Specification Observed value


1 Rizatriptan 4.6 1.00

2 Impurity C 7.0 9.1 1.52

3 Benzoic acid 12.9 2.80

S/N ratio of Sensitivity solution NLT 10 12.6

The benzoic acid retention time is somewhat larger than the specified value in the current monograph (and more than ± 10% of tabulated RRT). This deviation would most likely be approved benzoic acid is both well separated from the API and not interfering with the analysis of any other impurities

66

of any other impurities.


Chromatographic ConditionsColumn: Purospher® STAR Phenyl (2μm) Hibar® HR 100x2 1 mm 1 51014 0001

Rizatriptan and Related Substances (USP)Purospher® STAR Phenyl - UHPLC

Column: Purospher® STAR Phenyl (2μm) Hibar® HR 100x2.1 mm 1.51014.0001Injection: 2 μL (appropriate scaling per column tube volume reduction)Detection: UV 280 nmCell: 2.5 μLFlow Rate: 0.25 mL/min (not scaled according to linear velocity)Mobile Phase: A: Add 1.0 mL of trifluroacetic acid to 1 L of a solution of acetonitrile and water; 4:21 (v/v)

B: Acetonitrile and trifluroacetic acid 1000:1 (v/v) (or use our premixed product 4.80448)Gradient: See tableTemperature: 40˚CTemperature: 40 CDiluent: Solution ATest Solution: 1 mg/mL of USP Rizatriptan Benzoate System Suitability Mixture in Solution AResolution Solution: 0.5 μg/mL of Rizatriptan Benzoate obtained by suitable dilution of the Sample

solution with Solution APressure Drop: 205 - 195 Bar (2973 - 2828 psi)

150Time(min)

A(%)

B(%)

0.0 100 0

2.55 100 0

5.67 70 30

6.67 70 3060

90

120

150

ensi

ty (m

V)

C

6.7 100 0

10.0 100 0

0

30

60

0 2 4 6 8 10

Inte


Impu

rity

C



1 Rizatriptan 2.2 1.00

2 Impurity C 3.3 6.7 1.50

3


3 Benzoic acid 6.6 3.00

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Solvents and ReagentsProduct P/NAcetic acid (glacial) 100% anhydrous for analysis EMSURE® ACS,ISO,Reag. Ph Eur 1.00063

Acetonitrile isocratic grade for liquid chromatography LiChrosolv® 1.14291

Acetonitrile gradient grade for liquid chromatography LiChrosolv® Reag. Ph Eur 1.00030

Ammonia solution 28-30% for analysis EMSURE® ACS,Reag. Ph Eur 1.05423

Ammonium acetate for analysis EMSURE® ACS,Reag. Ph Eur 1.01116

Ammonium dihydrogen phosphate for analysis EMSURE® ACS,Reag. Ph Eur 1.01126

N,N-Dimethylformamide for analysis EMSURE® ACS,ISO,Reag. Ph Eur 1.03053

Ethanol gradient grade for liquid chromatography LiChrosolv® 1.11727

Hydrochloric acid fuming 37% for analysis EMSURE® ACS,ISO,Reag. Ph Eur 1.00317

Methanol for liquid chromatography LiChrosolv® 1.06018

Methanol gradient grade for liquid chromatography LiChrosolv® Reag. Ph Eur 1.06007

Octane-1-sulfonic acid sodium salt for ion pair chromatography LiChropur® 1.18307

ortho-Phosphoric acid 85% for analysis EMSURE® ACS,ISO,Reag. Ph Eur 1 00573ortho Phosphoric acid 85% for analysis EMSURE ACS,ISO,Reag. Ph Eur 1.00573

Perchloric acid 70% for analysis (max. 0.0000005% Hg) EMSURE® ACS,ISO,Reag. Ph Eur 1.00514

Potassium dihydrogen phosphate anhydrous 99.995 Suprapur® 1.05108

di-Potassium hydrogen phosphate trihydrate buffer substance for chromatography 1.19754

Potassium hydroxide pellets for analysis EMSURE® 1.05033

Sodium acetate anhydrous 99.99 Suprapur® 1.06264

Sodium dihydrogen phosphate dihydrate for analysis EMSURE® Reag. Ph Eur 1.06342

di-Sodium hydrogen phosphate dihydrate for analysis EMSURE® 1.06580

Sodium hydroxide pellets for analysis (max. 0.0002% K) EMSURE® ACS,Reag. Ph Eur 1.06495

Sodium perchlorate monohydrate EMSURE® 1.06564

Tetrahydrofuran LiChrosolv® 1.08101

Acetonitrile with 0 1% (v:v) trifluoroacetic acid for liquid chromatography LiChrosolv®Acetonitrile with 0.1% (v:v) trifluoroacetic acid for liquid chromatography LiChrosolv® 4.80448

Water for chromatography* 1.15333

* or use a Milli-Q Integral Water Purification System

Disclaimer: “Merck Millipore provide information and advice to our customers on application technologies and regulatory matters to the best of our knowledge and ability, but without obligation or liability. Existing laws and regulations are to be observed in all cases by our customers. This also applies in respect to any rights of third parties. Our information and advice do not relieve our customers of their own responsibility for checking the suitability of our products for the envisaged purpose Chromolith® Purospher®of their own responsibility for checking the suitability of our products for the envisaged purpose. Chromolith®, Purospher®, Emsure®, Lichropur®, Suprapur® and Lichrosolv® are all trademarks of Merck KGaA, Darmstadt, Germany.”

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Pharmacopoeia Monograph Methods

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